Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

Young, Jonathan W.; Locke, James; Altınok, Alphan; Rosenfeld, Nitzan; Bacarian, Tigran; Swain, Peter S.; Mjolsness, Eric; Elowitz, Michael B.

doi:10.1038/nprot.2011.432

Cited by 340 publications

(343 citation statements)

References 31 publications

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“…Cells also contained a fluorescent reporter of σ B activity. Strains were started from glycerol stocks and grown in Spizizen's minimal media (50) and prepared for microscopy using agarose pads (31) or analyzed using a Cellasic ONIX microfluidic platform with cells in logarithmic phase growth. For more details regarding the strain construction and growth please refer to SI Text.…”

Section: Methodsmentioning

confidence: 99%

Rate of environmental change determines stress response specificity

Young

Locke

Elowitz

2013

Proc. Natl. Acad. Sci. U.S.A.

119

142

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Cells use general stress response pathways to activate diverse target genes in response to a variety of stresses. However, general stress responses coexist with more specific pathways that are activated by individual stresses, provoking the fundamental question of whether and how cells control the generality or specificity of their response to a particular stress. Here we address this issue using quantitative time-lapse microscopy of the Bacillus subtilis environmental stress response, mediated by σ B . We analyzed σ B activation in response to stresses such as salt and ethanol imposed at varying rates of increase. Dynamically, σ B responded to these stresses with a single adaptive activity pulse, whose amplitude depended on the rate at which the stress increased. This rate-responsive behavior can be understood from mathematical modeling of a key negative feedback loop in the underlying regulatory circuit. Using RNAseq we analyzed the effects of both rapid and gradual increases of ethanol and salt stress across the genome. Because of the rate responsiveness of σ B activation, salt and ethanol regulons overlap under rapid, but not gradual, increases in stress. Thus, the cell responds specifically to individual stresses that appear gradually, while using σ B to broaden the cellular response under more rapidly deteriorating conditions. Such dynamic control of specificity could be a critical function of other general stress response pathways.systems biology | single-cell dynamics | computational biology C ells must respond to, and anticipate, a wide range of stresses that occur on multiple timescales. For this purpose, many species use general stress response pathways, which activate a diverse set of target regulons in response to a variety of stresses. For example, in mammals, p53 is activated by DNA damage (1-4) and hypoxia (5, 6), among others, and activates genes that impact cell cycle progression (7), DNA repair (8, 9), apoptosis, and angiogenesis (10, 11). In yeast, Msn2/4 responds to nutritional stress (12), as well as to salt (13), calcium (14), heat, and other stresses (15). Bacteria also contain general stress response pathways, including the alternative sigma factors RpoS in Escherichia coli (16) and σ B in Bacillus subtilis (17).It has been proposed that general stress response pathways enable cells to cross-protect, by anticipating stresses that may not be present at the moment, but are likely to occur soon (18). For example, preexposure to specific stresses is known to enhance bacterial resistance to different stresses applied subsequently (19)(20)(21). This raises a basic question: How do cells determine when to use the general stress response rather than activating more specific individual pathways?The σ B -mediated general stress response of B. subtilis provides an ideal model system to address these issues. σ B is activated by diverse stresses through a well-characterized and conserved transcriptional and posttranscriptional circuit mechanism (17). In response to stress, it activates ∼200 target ge...

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Section: Methodsmentioning

confidence: 99%

Rate of environmental change determines stress response specificity

Young

Locke

Elowitz

2013

Proc. Natl. Acad. Sci. U.S.A.

119

142

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“…Culture micrographs were automatically photographed at 10-or 15-min intervals were continuously acquired (Lin et al, 2010). Once all designed parameters have been confirmed, start time-lapse image observation (Young et al, 2012). Experiments were carried out three times for each condition.…”

Section: Time-lapse Imaging Analysismentioning

confidence: 99%

Alpha-lipoic acid-loaded nanostructured lipid carrier: sustained release and biocompatibility to HaCaT cellsin vitro

Wang

Xia²

2013

Drug Delivery

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ALA-loaded nanostructured lipid carrier (ALA-NLC) was designed to improve physicochemical stability and water solubility, and promote sustained release of ALA as well as determine the biocompatibility of ALA-NLC. The ALA-NLC manufactured using hot high-pressure homogenization technique was investigated in terms of size, zeta potential, FTIR analysis and release behavior. In vitro cytotoxicity and biocompatibility were determined by incubating with HaCaT cells using the MTT assay, HE staining and Hoechst 33342 staining. Cell behavior and cellular division of HaCaT cells untreated and treated by ALA-NLC were investigated in real-time images gathered using time-lapse imaging system. The release investigation illustrated that only 6.9% of ALA released in 30 min from ALA-NLC formation, whereas it was 30.3% in free ALA system. ALA-NLC possessed a satisfactory release behavior of sustained release up to 72 h. It showed that ALA-NLC did not exert hazardous effect on HaCaT cells up to 81.9 mg/L without morphological alterations, revealing a satisfactory biocompatibility. Evidence was provided from time-lapse imaging system that cell behavior and cellular division of ALA-NLC treated HaCaT cells were in accordance with the control. These results of this investigation demonstrated that NLC encapsulated ALA formation (ALA-NLC) can improve stability, solubility and release of ALA; ALA-NLC was biocompatible to HaCaT cells.

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“…It enables time-lapse investigations of microcolonies with single cell resolution, however, at very low throughput and minimal environmental control. 16 To overcome some of these limitations, automated microscopy and image recognition software have been applied to analyse multiple bacteria microcolonies on a single agar pad. 17 Nevertheless, lack of environmental and spatial cell growth control remain major drawbacks.…”

Section: Introductionmentioning

confidence: 99%

A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level

et al. 2012

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In the continuously growing field of industrial biotechnology the scale-up from lab to industrial scale is still a major hurdle to develop competitive bioprocesses. During scale-up the productivity of single cells might be affected by bioreactor inhomogeneity and population heterogeneity. Currently, these complex interactions are difficult to investigate. In this report, design, fabrication and operation of a disposable picolitre cultivation system is described, in which environmental conditions can be well controlled on a short time scale and bacterial microcolony growth experiments can be observed by time-lapse microscopy. Three exemplary investigations will be discussed emphasizing the applicability and versatility of the device. Growth and analysis of industrially relevant bacteria with single cell resolution (in particular Escherichia coli and Corynebacterium glutamicum) starting from one single mother cell to densely packed cultures is demonstrated. Applying the picolitre bioreactor, 1.5-fold increased growth rates of C. glutamicum wild type cells were observed compared to typical 1 litre labscale batch cultivation. Moreover, the device was used to analyse and quantify the morphological changes of an industrially relevant L-lysine producer C. glutamicum after artificially inducing starvation conditions. Instead of a one week lab-scale experiment, only 1 h was sufficient to reveal the same information. Furthermore, time lapse microscopy during 24 h picolitre cultivation of an arginine producing strain containing a genetically encoded fluorescence sensor disclosed time dependent single cell productivity and growth, which was not possible with conventional methods.

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Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy

Cited by 340 publications

References 31 publications

Rate of environmental change determines stress response specificity

Rate of environmental change determines stress response specificity

Alpha-lipoic acid-loaded nanostructured lipid carrier: sustained release and biocompatibility to HaCaT cellsin vitro

A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level

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